A factor which became familiar as tumor necrosis factor (TNF) was first encountered by Carswell, et al, Proc Natl Acad Sci (USA) (1975) 72:3666. It was found that the sera of endotoxin treated mice, rabbits, or rats which had been previously sensitized with an immunopotentiator such as Bacillus Calmette-Gurin (BCG) contained a substance which, when injected into mice harboring transplanted tumors, caused extensive hemorrhaging of the tumors, without undesirable side effects on the recipient. The sera were thus presumed to contain a substance which was selectively necrotic to tumor cells, and neutral with respect to its reactions with normal tissue, hence the designation, TNF. The ability to cause this selective tumor destruction when injected into whole animals became a standard in vivo assay defining TNF.
TNF has also been produced in cell culture. Matthews, et al, Brit J Cancer (1981) 44:418 were able to obtain TNF activity in the medium of mononuclear phagocytes derived from BCG-injected rabbits; Mannel, et al, Infect Immun (1980) 30:523; ibid (1981) 33:156 obtained TNF activity from the medium of macrophage-enriched peritoneal exudate cells from BCG-infected mice after the cell culture was induced with endotoxin.
Attempts have been made to purify whatever factor is responsible for the selective cytotoxicity against neoplastic cells but, because the substances are apparently present only in tiny amounts either in the serum of whole animals or in tissue culture media, it has not been possible to effect complete purification. Furthermore, the protein or proteins are evidently unstable, and two recent U.S. patents, Nos. 4,447,355 and 4,457,916 are directed to methods for stabilizing the activity of the preparation by addition of, for example, albumin or a carbohydrate material. In the procedures of those disclosures, using standard purification procedures developed by others, it was possible to obtain a specific activity for preparations of TNF to approximately 1.times.10.sup.6 units/mg, where units were defined in terms of an in vitro assay for cytotoxicity against murine L-M cells (ATCC CCL 1.2). It has not been possible, however, to obtain material which is both active in the in vivo (Carswell) tumor necrosis assay for TNF and of sufficient purity to permit amino acid sequence information to be obtained.
Indeed, because of the unavailability of pure cytotoxic protein, it is unclear at present how many proteins may be available which are selectively necrotic to cancer cells. The in vivo method of Carswell, et al (supra) has been accepted as the standard defining TNF. Because of the cross species activity of these factors, this assay is, in one sense, conveniently diagnostic. However, the more conveniently performed in vitro assay for cytotoxicity has frequently been used as an index of TNF activity, despite considerable confusion about whether there exists a one-to-one correlation between the in vitro assay and the test defining TNF. Indeed, a protein derived from a transformed B-cell line, which is active in the in vitro assay, has been designated "lymphotoxin", purified to homogeneity and partially sequenced (Genentech, EPO Patent Publication 0100641, published Feb. 15, 1984). It has been assumed that lymphotoxin is a different protein from "TNF" because it is of non-macrophagic origin. Further, anti-sera prepared agasint lymphotoxin do not cross react with the cytotoxic (TNF) factor purified from macrophage (Stone-Wolff, D., et al, J Exp Med (1984) 159:828.
Provision of a defined protein sequence which is capable of a cytotoxic effect specifically directed against tumor cells would, of course, represent a major benefit for both diagnosis and therapy of malignant diseases. It appears, also, that certain of these factors may exhibit anti-parasitic activity; it has been shown that a protein designated TNF, derived from sera of BCG injected mice, exhibits cytotoxic affects on malaria parasites (Plasmodium falciparium)) in vivo and in vitro (Haidans, et al, Infect Immun (1983) 42:385).